Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, F-31077, Toulouse, France.
Molecular Forces Consulting, 40 rue Boyssonne, F-31400, Toulouse, France.
Sci Rep. 2019 Aug 9;9(1):11576. doi: 10.1038/s41598-019-48091-7.
In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.
在这项工作中，我们描述了一种合成代谢途径的构建，该途径能够通过柠檬酸循环中间体苹果酸将葡萄糖直接合成为 1,3-丙二醇（PDO）。这条非天然途径通过三个额外的反应步骤扩展了先前发表的从苹果酸合成（L）-2,4-二羟基丁酸（L-DHB）的合成途径，这三个反应步骤分别由 DHB 脱氢酶、2-酮-4-羟基丁酸（OHB）脱氢酶和 PDO 氧化还原酶催化。筛选和结构导向的蛋白质工程提供了一种（L）-DHB 脱氢酶，它来自大肠杆菌的膜结合（L）-乳酸脱氢酶，以及 OHB 脱羧酶变体，这些变体来自乳球菌属的分支链酮酸脱羧酶编码的 kdcA 或运动发酵单胞菌的丙酮酸脱羧酶。这些酶的基因同时过表达，再加上内源性 ydhD 编码的醛还原酶，使得（L）-DHB 能够合成 PDO。虽然这六种酶的活性在一个工程大肠杆菌菌株中同时表达导致从 110mM 葡萄糖中仅产生 0.1mM 的 PDO，但通过共培养表达苹果酸-DHB 途径的大肠杆菌菌株和另一个携带 DHB 到 PDO 途径的菌株，PDO 的产量增加了 40 倍。
